Effect And Mechanism Of Salidroside On Palmitic Acid Transportation Across Vascular Endothelial Cells And Myocardial Insulin Resistance Induced By TNF-?

Objective:Salidroside is the main biological active ingredient of Rhodiola rosea.Studies have shown that salidroside can treat Type 2 Diabetes?T2DM?by improving insulin resistance?IR?.Tumor necrosis factor-??TNF-??can promote the transport of palmitic acid?PA?across vascular endothelial cells and aggravate PA-induced myocardial insulin resistance.The aim of this study is to investigate the effect and mechanism of salidroside on TNF-?promoting PA transportation across microvascular endothelial cells and promoting myocardial insulin resistanceMethods:?1?The effect of salidroside and caspase-1 inhibitor?Ac-YVAD-cmk?on the TNF-?-promoting fatty acids across microvascular endothelial cells was detected by using fatty acid in-vitro transcytosis model.?2?The expression of NLRP3,pro-caspase-1,pro-interleukin-1??pro-IL-1??,caspase-1 and Interleukin-1??IL-1??were detected by Western blotting and the effect of salidroside on TNF-?-activated NLRP3 inflammasome was evaluated.?3?Using 2,7-dichlorodi-hydroflurescein diacetate?DCFH-DA?and MitoSOX^TMM Red fluorescent probes indicating cytoplasmic ROS and mitochondrial superoxide anion levels respectively,the effect of salidroside on TNF-?-promoted oxidative stress in CMECs was evaluated.?4?In the co-culture model of neonatal rat cardiomyocytes and CMECs,insulin-stimulated 2-NBDG uptake by cardiomyocytes was measured and the effect of salidroside on TNF-?aggravating PA-induced insulin resistance in cardiomyocytes was determined.?5?In the animal experiment,C57BL/6J mice were treated with TNF-?,PA,and or SAL,respectively.Using a small animals positron emission tomography?PET?,insulin-stimulated myocardial¹⁸F-FDG uptake was detected and the percent injection dose rate?%ID/cc?,were calculated.After PET scanning,the mice were sacrificed,the hearts were collected,and cardiac emission values were directly measured using a gamma counter.Results:?1?TNF-?increased the transportation of fatty acids across CMECs using the in-vitro fatty acid transcytosis model,and salidroside could inhibit TNF-?-promoted fatty acids transcytosis.The caspase-1 inhibitor?Ac-YVAD-cmk?also inhibited TNF-?-promoted fatty acids transcytosis.Compared with Ac-YVAD-cmk alone,co-incubation with Ac-YVAD-cmk and salidroside didn't show synergy.?2?Western Blotting results showed that TNF-?significantly increased the expression of NLRP3,pro-caspase-1,caspase-1 and IL-1?,while salidroside significantly inhibited the expression of NLRP3 and IL-1?induced by TNF-?.?3?By using DCFH-DA fluorescent probe,TNF-?-caused a time-dependent increase in ROS levels in CMECs was observed;and salidroside could inhibit TNF-?-induced increase in ROS levels.Similar phenomenon were found in mitochondrial superoxide anion levels detected by using MitoSOX^TM Red.Under incubation of TNF-?,mitochondrial superoxide anion level in CMECs increased with increasing TNF-?incubation time and salidroside blocked this effect.?4?In in-vitro CMECs-cardiomyocyte co-culture experiment,TNF-?aggravated PA-induced insulin resistance in cardiomyocytes;while salidroside inhibited this effect.?5?Small animals PET results showed that under insulin stimulation,the mice in the group of TNF-?+PA had less uptake of ¹⁸F-FDG.Salidroside improved insulin-induced ¹⁸F-FDG uptake.The results of the cardiac gamma counter test were consistent with the PET scan results.Conclusion:TNF-?up-regulates ROS in cytoplasmic and superoxide anion in mitochondria and activates NLRP3 inflammasome in CMECs.Activation of NLRP3 inflammasome is involved in TNF-?promoting PA transcytosis across CMECs,thereby aggravating PA-induced insulin resistance.Salidroside may play a role in inhibiting TNF-?-promoted fatty acid transcytosis and insulin resistance by reducing oxidative stress and NLRP3 activation induced by TNF-?.